Antiviral Resistance in Hepatitis B

SILS 2009 - The 5th Seoul International Liver Symposium - Symposium Ⅱ B r e a k i n g t h e b r i c k s i n h e p a t i t i s B t r e a t m e n t Antiviral resistance in hepatitis B Stephen Locarnini Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia = Abstract = A high rate of viral turnover, combined with an error‐prone polymerase, results in an increased frequency of mutational events during hepatitis B virus (HBV) replication, resulting in a diverse population of progeny virus (quasispecies). Not suprising then, particular selection pressures, both from within (host immune clearance) or from outside (vaccines and anti- virals) the host, readily select out new “escape” mutants resulting in treatment failure. The introduction of nucleos(t)ide analogue (NA) therapy for chronic hepatitis B has resulted in the emergence of antiviral drug resistance which has itself become the major factor limiting treatment effectiveness. Furthermore, due to the overlap of the viral polymerase and en- velop reading frames in the circular HBV DNA genome, NA‐resistance associated mutations selected within the catalytic domains of the polymerase usually result in significant changes to the neutralising antibody binding domains of the hepatitis B surface antigen, including the emergence of antiviral drug associated potential vaccine escape mutants (ADAPVEM’s). The main reason for this is that the neutralisation domain, the “a” determinant, is a conformational epitope. The public health significance of APADVEM’s may then be very considerable in terms of the global program for control of hepatitis B via universal infant immunisation. Thus, prevention of resistance requires the adoption of strategies that not only effectively control active HBV replication but also prevent the emergence of APADVEMs. Introduction to antiviral drug resistance Two key concepts are critical to an understanding of the development of antiviral drug resistance. First, viral infection is typically characterised by high levels of viral production and turnover. Second, the viral population in an infected person is highly heterogeneous. In an infected individual, there is a cycle of “viral reproduction”, viral mutant generation, genetic diversity, antiviral drug selection pressure leading to “survival of the fittest”, directly as a consequence of quasispecies dominance.1 Antiviral drug resistance also depends on at least six factors: 1. Viral mutation frequency. 2. The magnitude and rate of virus replication. 3. Intrinsic mutability of the antiviral target site (usually a viral enzyme). 4. Selective pressure (potency) of the drug. 31 SILS 2009 - The 5th Seoul International Liver Symposium - 5. Amount of replication space. 6. Fitness of the resistant mutant. Other factors that can play a role include the “genetic barrier” of the drug which can be considered in the context of the number of specific mutations required for drug resistance to develop.2,3 One of the major classes of antiviral agent is the nucleos(t)ide analogues (NA), which are competitive in- hibitors of the viral DNA polymerase enzyme and most NAs block viral replication by premature chain termi- nation, since they lack a 3’‐OH group.4 This class of drug has been extensively studied in both HIV as well as HBV‐disease, and several mechanisms of NA resistance have been identified.2,4,5 1. Steric hindrance where the associated resistance substitution alters the ability of the viral enzyme to bind NA relative to the natural substrate. 2. Reduction in catalytic efficiency where the resistance substitution results in sub‐optimal nucleophilic attack geometry for the subsequent information of NA into the newly replicating viral genome. 3. Increased excision of the NA as chain terminator by the process of pyrophosphorolysis. By understanding the main processes involved in selection of drug resistant viruses, it is possible then to implement ways to prevent it:3 1. Maximise antiviral activity. 2. Maximise genetic barriers to resistance. 3. Maximise pharmacologic barriers. Thus in this way, the emergence of resistant viruses should be minimised. In order to overcome resistance, the best approach has been to use combination chemotherapy; in hepatitis B, on first virological breakthrough, an add‐on strategy using an agent with a complementary cross‐resistance profile is the preferred approach.6 HBV virology and pathways to resistance The lifecycle of the hepatitis B virus (HBV) revolves around two key processes: (a) generation of HBV co- valently closed circular (ccc) DNA from genomic relaxed circular (RC) DNA and its subsequent processing by host enzymes to produce viral RNA; and (b) reverse transcription of the pregenomic (pg) RNA within the viral nucleocapsid to form RC DNA, thereby completing the viral life cycle. In the patient on a daily basis, the replication phases of HBV are marked by a high frequency of mutational events resulting from an enormous viral turnover rate combined with the error‐prone reverse transcriptase/polymerase, producing a quasispecies pool comprising a particular HBV population that is dominant at any one point in time.1 Not surprisingly then, the introduction of nucleos(t)ide analog (NA) therapy has seen the emergence of antiviral drug resistance, which has become the main factor limiting the long‐term application of antiviral agents such as NA for patients with chronic hepatitis B (CHB). To date, there are eight codons associated with primary antiviral drug resistance in CHB, which map to four 32 SILS 2009 - The 5th Seoul International Liver Symposium - of the functional domains of the HBV Pol7,8 (a) B‐Domain at Codons 169, 180, 181, 184; (b) C‐Domain at Codons 202 and 204; (c) D‐Domain at Codon 236; and (d) E‐Domain at Codon 250. Several major HBV evolutionary NA‐resistance pathways (rtM204I/V, rtN236T and rtA181T/V) have now been characterised associated with these eight codons.8 The first or rtM204V/I pathway is responsible for resistance to the L‐nucleosides such as lamivudine (LMV) and telbivudine (LdT), and also entecavir (ETV) as rescue therapy in LMV‐experienced patients. The L‐nucleoside pathway is associated with clusters of secondary mutations that can affect subsequent treatment with NAs (rtT184G, rtS202I) such as ETV. The second or the rtN236T pathway, accounts for adefovir (ADV) and tenofovir (TFV) resistance. The third pathway, rtA181T/V, is associated with resistance to LMV and ADV and is a potential multi‐drug resistance (MDR) pathway and will probably impact on TFV sensitivity, as well, either alone or with the rtN236T. In naïve patients only treated with ETV, a fourth pathway has been described where at least 3 mutations are need to be selected out at the same time: rtL180M+rtM204V plus either one of rtT184 or rtS202 or rtM250 codon changes. Finally, in highly experienced NA treated patients, other MDR pathways are being increasingly recognised such as rtA181T+rtN236T+rtM250L. Sequential monotherapy treatment with NAs promotes multi‐drug resistance. Thus, the prevention of resistance will require the adoption of strategies that effectively control virus replication and exploit an understanding of the mechanisms and processes that drive the emergence of drug resistance namely, high replication rates, low fidelity of the HBV reverse transcriptase/polymerase, selective pressure of the NA, genetic barrier of the drug, role of replication space (liver turnover) and fitness of the mutant.7 Cross‐resistance Cross resistance is defined as resistance to drug(s) to which a virus has never been exposed.2 The NA resistance‐associated mutations selected by particular groups of NA (eg: L‐nucleosides, Acyclic Phosphonates or D‐ Cyclopentane) may diminish the antiviral activity of other drugs.6 This should be considered before any antiviral drug is prescribed and the physician should plan for eventual treatment failure. The initial selection and subsequent rescue therapies should be based on a knowledge of cross‐resistance,6 so that the second agent lacks cross‐resistance with the failing agent.9,10 Preferably by using the add‐on/combination approach9,10 the advantage of using combinations of NA with complementary cross‐resistance profiles has recently been highlighted6 and a summary of cross‐resistance profiles based on the viral resistance “pathways” approach is shown in Table 1. Public health relevance of resistance The viral envelope (HBsAg) gene overlaps completely within the reverse transcriptase gene and so NA resistance can result in changes in HBsAg. This Pol‐Env overlap is important for a number of reasons since it 33 SILS 2009 - The 5th Seoul International Liver Symposium - Table 1. Cross-resistance analysis for the nucleos(t)ide analogues approved for chronic hepatitis B* Resistance mutation* LVD/LdT-resistant ADV-resistant ADV-resistant ETV-resistant LdT-resistant (L180M +/- M204V/I) (N236T) (A181T/V) Mutation confers some • Entecavir • Tenofovir • Lamivudine/ • Entecavir degree of reduced Telbivudine sensitivity to listed drugs • Tenofovir Mutation confers • Telbivudine • Lamivudine • Lamivudine complete resistance • Telbivudine Drugs remaining fully • Adefovir • Entecavir • Entecavir • Adefovir • Adefovir active • Tenofovir • Lamivudine • Tenofovir • Tenofovir • Telbivudine *Modified from References 6 & 7. *First virological breakthrough should be managed with an add-on strategy (combination), not switch (sequential monotherapy). has been shown that common LMV resistant HBVs such as (rtV173L+rtL180M+rtM204V) have important and significant changes in HBsAg (sE164D+sI195M) which significantly reduce anti‐HBs (vaccine‐associated) binding in vitro. Likewise, in ADV failure, the rtA181T HBV can be found either by itself or in association with rtN236T, in up to 40% of cases. The rtA181T in rt results in a sW172 [stop] in the overlapping HBsAg and this mutant is defective in virion secretion, is retained in the cell, and acts as a dominant negative mutant for wild‐type HBV secretion.11 The clinical implication of these observations is that the virological case definition of drug resistance, >1.0 log IU/mL from nadir in two consecutive samples taken 1 month apart,9,10 does not apply if this mutant is (co)‐selected.

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